Thread by @mad_lancaster

I'm thrilled to share our latest published paper in @eLife
where we applied cryo-EM to brain organoids to look at ultrastructure of human axons with unprecedented resolution!

Check out the lovely cryo-CLEM clip below.
And a short 🧵 of what we found.

elifesciences.org/articles/70269

First off, we established a method to culture our air-liquid interface organoid cultures with EM grids to get outgrowth of axon bundles onto the grids. This enables capture of "clean" axons without dendrites like you normally get with cells in vitro.

Then, using correlative light and electron microscopy (CLEM) we could trace axon bundles and focus in on GFP labeled axons within bundles to explore their intracellular architecture.

We found that human growing axons are really unique! For example we know that microtubules are parallel and unidirectional in axons, and we could see that, but we even had the resolution to do subtomogram averaging and see individual protofilaments.

We also found some really interesting ER morphologies that seem to be unique to axons, with incredibly thin tubules almost completely lacking lumen, pointing to a primary role in lipid biosynthesis in this context, which makes sense given the huge surface area of axons.

Finally, we were surprised by the scarcity of ribosomes specifically in the axon shaft, a finding previously seen in more traditional EM, but now corroborated with cryo-EM which has the resolution to pick up even monosomes if they are there.

This has implications in terms of protein biogenesis, and suggests that local translation is not a major contributor along the length of the axon. Importantly, other neuronal processes (i.e. dendrites) had plenty of ribosomes, and we did not capture synapses or growth cones.

We hope that this big dataset of tomograms from human axons will be a useful resource for the community. Explore for yourself and access the full dataset on EMDB and EMPIAR (accession codes in the paper).

This was an awesome collaboration between my lab and @KukulskiWanda. It was the serendipitous result of our labs being next door, and many fruitful lunch breaks with co-first authors Patrick Hoffmann and Stefano Giandomenico. It helps to have friends that can do amazing science!!